The present invention relates to certain compounds, especially pyrimidine derivatives, which show activity in biological systems as cyclin dependent kinase (CDK) inhibitors and which are accordingly of interest as potentially useful therapeutic agents that may be incorporated in pharmaceutical compositions or formulations for use in controlling or inhibiting cell growth or proliferation in mammals, for example in connection with antitumour or cancer treatment.
Cyclin dependent kinases (CDK""s) are a family of enzymes which form complexes with other activating proteins known as cyclins to provide key regulatory factors that are involved in the control of growth and division in animal cells. More particularly, the progression of animal cells through the cell division cycle (G1, S, G2 and M phases) is regulated by the sequential formation, activation and subsequent inactivation of a series of CDK/cyclin dimer complexes which control passage past cell cycle checkpoints and transitions between successive phases of the cell cycle, with the CDK""s acting as catalytic sub-units of the complexes.
There are in fact a number of different cyclin proteins which, like the different CDK""s, form a somewhat loosely related family of CDK-activating proteins; different CDK/cyclin complexes function at different stages of the cell cycle with sequential increase and decrease in cyclin expression during the cell cycle and cyclin degradation during M phase usually being an important factor in determining orderly cell cycle progression. Thus, progression through G1 to S phase in mammalian cells is believed to be regulated primarily by cyclin dependent kinases CDK2, CDK3 and CDK4 (and possibly also CDK6 in some cells) in association with at least cyclins D and E, the complexes of CDK2 and CDK4 (and possibly CDK6) with D type cyclins in particular playing an important role in controlling progression through the G1 restriction point whilst the CDK2/cyclin E complexes are essential for bringing about the transition from G1 into S phase. Once S phase is entered it is believed that further progression and entry into G2 then requires activated complexes of CDK2 with another cyclin which is designated cyclin A, i.e. complexes CDK2/cyclin A. Finally, for the transition from G2 phase to M phase and initiation of mitosis, activated complexes of the cyclin dependent kinase designated CDK1 (also known as Cdc2) with a cyclin designated cyclin B (and also complexes of CDK1 with cyclin A) are required.
In general, control of the cell cycle and activity of CDK""s involves a series of stimulatory and inhibitory phosphorylation and dephosphorylation reactions, and in exercising their regulatory functions the CDK/cyclin complexes when activated use ATP as a substrate to phosphorylate a variety of other substrate cell proteins, usually on serine and threonine groups thereof. Control of the cell cycle may also involve inhibitors of CDK/cyclin complexes which block the catalytic function of these enzymes so as to lead to arrest of the cell cycle. Certain natural inhibitors, such as for example the inhibitory proteins known as p16 and p21, can block cell cycle progression by binding selectively to CDK/cyclin complexes to inactivate the latter.
Control by inhibitors of CDK function may therefore provide a further mechanism for controlling cell cycle progression, and this has led to proposals for using CDK inhibitors as antiproliferative therapeutic agents, in antitumour therapy for example, for targeting abnormally proliferating cells and bringing about an arrest in cell cycle progression. This has seemed to be especially appropriate since it is known that severe disorders or irregularities in cell cycle progression frequently occur in human tumour cells, often accompanied by over-expression of CDK""s and other proteins associated therewith. Also, compared to established cytotoxic antitumour drugs, the use of inhibitors of cell proliferation acting through CDK""s would have the advantage of avoiding a direct interaction with DNA, thereby giving a reduced risk of secondary tumour development.
The potential therapeutic applications and other possible uses have accordingly led to a search for further chemical inhibitors of CDK""s, especially selective inhibitors that may be suitable for pharmaceutical use. Inhibitory activity and selectivity of selected CDK/cyclin complexes is generally assayed by measuring the kinase activity in phosphorylating the protein histone H1 (one of the major protein constituents of chromatin which generally provides a good CDK substrate) in the presence of the suspected inhibitor under test. A number of compounds having potentially useful CDK inhibitory properties that have been identified in this way are described in a review article, of which the content is incorporated herein by reference, entitled xe2x80x9cChemical inhibitors of cyclin-dependent kinasesxe2x80x9d by Laurent Meijer published in Cell Biology (Vol. 6), October 1996. Among the compounds referred to in the above-mentioned article is a potent CDK1 and CDK2 inhibiting adenine derivative 2-(2-hydroxyethylamino)-6-benzylamino-9-methyl-purine, named xe2x80x9colomoucinexe2x80x9d, and also a close analogue incorporating modifications at positions 2, 6 and 9, namely, 6-(benzylamino)-2(R)-[{1-(hydroxymethyl)propyl}amino]-9-isopropylpurine. This latter compound is named xe2x80x9croscovitinexe2x80x9d and is even more potent than olomoucine as a CDK inhibitor. The strong but selective CDK inhibitory properties of olomoucine were first described in a paper by J. Vesely et al entitled xe2x80x9cInhibition of cyclin-dependent kinases by purine analoguesxe2x80x9d, Eur. J. Biochem. 224, 771-786 (1994), and further studies on CDK inhibitory properties of a range of purine compounds in the form of adenine derivatives, including olomoucine and roscovitine, are reported and discussed in a paper by L. Havlicek et al entitled xe2x80x9cCytokinin-Derived Cyclin-Dependent Kinase Inhibitors: Synthesis and cdc2 Inhibitory Activity of Olomoucine and Related Compoundsxe2x80x9d J. Med. Chem. (1997) 40, 408-412. Again, the content of these publications is to be regarded as being incorporated herein by reference.
The inhibitory activity of both olomoucine and roscovitine has been shown to result from these compounds acting as competitive inhibitors for ATP binding. It may be noted that olomoucine at least is reported as having a total lack of inhibitory activity in relation to many common kinases other than CDK""s. Selectivity is further manifest by the fact that both olomoucine and roscovitine inhibit activity of CDK1, CDK2 and CDK5, but neither has been found to be active against CDK4 or CDK6.
Olomoucine in particular has been regarded as providing a lead compound for helping to identify and design further purine based CDK inhibitors, and based on structure/activity studies it was suggested in the above-mentioned paper of Vesely et al that N9 substitution by a hydrophobic residue such as methyl, 2-hydroxyethyl or isopropyl was important, e.g. to provide a direct hydrophobic interaction with the CDK, and that a side chain at C2 appeared to be essential. Similarly, in the paper of Havlicek et al, apart from observing that for purine compounds to have CDK inhibitory activity the 1 and 7 positions, and possibly the 3 position, of the purine ring must remain free to permit hydrogen bonding, it was also stated that a polar side chain at position 2 appears to be essential and that N9 substitution by a hydrophobic residue is also probably important for positive binding. Positions 2, 6 and 9 in the purine ring were identified as being the positions which control to CDK1.
In the review article of Meijer, it is also mentioned that as a result of crystallization of CDK-inhibitor complexes, and in particular co-crystallization studies with CDK2, it has been found that inhibitors such as olomoucine and roscovitine localize in the ATP binding pocket which is located in the cleft between the small and large lobes of the CDK protein molecule, and that specificity was probably provided by portions of the inhibitor molecules interacting with the kinases outside the ATP binding sites.
The present invention has developed from an observation made in the course of testing various guanine derivatives for activity as inhibitors of the DNA repair protein O6-methylguanine DNA-methyltransferase (MGMT) when it was found unexpectedly that although the compound O6-cyclohexylmethylguanine had very little activity as a MGMT inhibitor, it was nonetheless cytotoxic and showed very high inhibitory activity, comparable to that of olomoucine, against CDK1(cdc2)/cyclin B complexes. This was particularly surprising against the background discussed above in relation to olomoucine given that this guanine compound has no substituents at either the 2-NH2 position or the 9 position in the purine ring and that the replacement of the 6-NH by 6-O made the compound less like ATP with which olomoucine at least is believed to compete for binding sites.
Subsequently, other guanine derivatives have been identified, more closely related to O6-cyclohexylmethylguanine than to compounds such as olomoucine and roscovitine, which show significant CDK inhibitory activity, and crystallographic studies have revealed that complexes of CDK2 (homologous with CDK1, at least in respect of the catalytic binding site) with guanine derivatives such as O6-cyclohexylmethylguanine and O6-cyclohex-1-enylmethylguanine bind together in a different manner from complexes of CDK2 with olomoucine.